All aspects of transcription are controlled by epigenetic chromatin alterations that occur at the level of individual genes, gene clusters, or whole chromosomes. Transcription relies on the assembly of the pre- initiation complex and its subsequent activation; yet it is now abundantly clear that the elongation stage of the transcription cycle, with its coordinated transcript-processing events, plays a major role in the regulation of gene expression. In spite of an amazing level of progress, the reality remains that we still do not understand many aspects of the targeting of complexes that modify or remodel chromatin, the dynamics of their association with their targets and the molecular basis of the mechanistic consequences of this association on the process of transcription. This may be particularly true of complexes and their epigenetic modifications that do not promote the initiation or repression of transcription but rather regulate the level of gene expression. The model system that we have been studying - the MSL complex responsible for the phenotypic phenomenon of dosage compensation in Drosophila - is an example of this latter type of complexes that operate at the whole-chromosome level on the transcription of individual genes. The function of the MSL complex is unlikely to be the initiation of gene activity; rather we favor the hypothesis that the enhancement in the level of gene expression that leads to dosage compensation is the result of an enhancement in the rate of transcription elongation. Our goal is to understand the mechanism of action of the MSL complex at the molecular and biophysical levels and thereby contribute to our general understanding of the role that epigenetic modifications play in the all-important elongation process. Towards this end, we propose to use (1) traditional molecular cytology in transgenic flies, (2) an experimental system that recapitulates dosage compensation on plasmids in transfected cells, and (3) single-molecule assays. We will make every effort to relate our in vitro results with in vivo phenotypes in order to understand the underlying cellular mechanisms. Related complexes exist in yeast and we have identified and characterized a homologous complex in humans that carries out the same histone modification as the Drosophila complex. PUBLIC HEALTH RELEVANCE: In cells, DNA is associated with octamers of basic proteins, the histones - this association is unfavorable to the expression of genes and must be modified - the modifications that allow genes to be expressed, or that can be reversed to shut down genes, are called epigenetic modifications since they do not occur in the DNA. Errors in epigenetic modifications have been correlated to various developmental defects, metabolic diseases and cancers. We are studying a specific modification that affects the expression of genes on one of the sex chromosomes of the fruitfly Drosophila in order to uncover some general principles underlying epigenetic regulation; significantly, we have discovered that the same type of modification occurs in humans where it affects the expression of genes on all of the chromosomes.